Adeno-associated virus (AAV) is a human DNA virus which contains a linear, single-stranded, DNA genome, approximately 5kb long. One of the unusual aspects of AAV is the mechanism of DNA replication, which involves only leading strand DNA synthesis. Viral replication relies on the AAV coded rep gene and cellular replication enzymes. Therefore, studies of AAV DNA replication should be useful for identifying the cellular enzymes that are involved uniquely in leading strand synthesis. During DNA replication, the virus uses a 145 bp hairpinned terminal repeat to prime DNA synthesis, and then regenerates the primer by a process called terminal resolution. In addition to being the origins for DNA replication, the terminal repeats also contain sequences that are essential for viral packaging and for integration of the viral genome into host chromosomes. In our previous work we purified the AAV Rep68 and 78 proteins and demonstrated that they are the site specific endonucleases that initiate terminal resolution. We also showed that they have an intrinsic DNA helicase activity. During the course of this work we developed several biochemical assays to study the interaction of Rep with the terminal repeats. These include the terminal resolution assay and the site specific nicking assay (or trs endonuclease reaction). More recently, we have also developed a complete in vitro DNA replication assay. Using these as well as conventional binding assays we demonstrated that there are at least three separate sequences within the terminal repeat that are required for substrate recognition by the Rep protein: the secondary structure element, a linear Rep binding sequence, and a specific sequence required for nicking by the enzyme, the terminal resolution site (trs). Finally, we and others have shown that the Rep proteins have multiple roles during the viral life cycle. In addition to being essential for DNA replication, Rep68 and 78 are also required for transactivation of AAV transcription, repression of AAV and heterologous gene expression, and the repression of oncogenic transformation by some viral and cellular oncogenes. This suggests that the Rep proteins probably interact with a variety of cellular proteins. Our specific aims are: 1) To characterize in detail the three elements of the terminal repeat required for origin function. In the process we also hope to identify sequences in the terminal repeat that are required for integration and packaging. 2) To purify and identify the cellular DNA replication enzymes that are required for AAV DNA replication. 3) To identify the cellular enzymes that interact with the AAV Rep protein.